Enzyme-substrate Interactions Research Articles

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve.

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1min vs. 36h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6's large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde's spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

Hydrolytic enzyme activity in high-rate anaerobic reactors treating municipal wastewater in temperate climates

Particulate matter hydrolysis is the bottleneck in anaerobic treatment of municipal wastewater in temperate climates. Low temperatures theoretically slow enzyme-substrate interactions, hindering utilization kinetics, but this remains poorly understood. β-glucosidase, protease, and lipase activities were evaluated in two pilot-scale upflow anaerobic sludge blanket (UASB) reactors, inoculated with different sludges and later converted to anaerobic membrane bioreactors (AnMBRs). Despite similar methane production and solids hydrolysis rates, significant differences emerged. Specific activity peaked at 37 °C, excluding the predominance of psychrophilic enzymes. Nevertheless, the Michaelis-Menten constant (Km) indicated high enzyme-substrate affinity at the operational temperature of 15–20 °C, notably greater in AnMBRs. It is shown, for the first time, that different seed sludges can equally adapt, as hydrolytic enzymatic affinity to the substrate reached similar values in the two reactors at the operational temperature and identified that membrane ultrafiltration impacted hydrolysis by a favourable enzyme Michaelis-Menten constant.

Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations.

Methyl-parathion hydrolase (MPH), which evolved from dihydrocoumarin hydrolase, offers one of the most efficient enzymes for the hydrolysis of methyl-parathion. Interestingly, the substrate preference of MPH shifts from the methyl-parathion to the lactone dihydrocoumarin (DHC) after its mutation of five specific residues (R72L, L273F, L258H, T271I, and S193Δ, m5-MPH). Here, extensive QM/MM calculations and MM MD simulations have been used to delve into the structure-function relationship of MPH enzymes and plausible mechanisms for the chemical and nonchemical steps, including the transportation and binding of the substrate DHC to the active site, the hydrolysis reaction, and the product release. The results reveal that the five mutations remodel the active pocket and reposition DHC within the active site, leading to stronger enzyme-substrate interactions. The MM/GBSA-estimated binding free energies are about -20.7 kcal/mol for m5-MPH and -17.1 kcal/mol for wild-type MPH. Furthermore, this conformational adjustment of the protein may facilitate the chemical step of DHC hydrolysis and the product release, although there is a certain influence on the substrate transport. The hydrolytic reaction begins with the nucleophilic attack of the bridging OH- with the energy barriers of 22.0 and 18.0 kcal/mol for the wild-type and m5-MPH enzymes, respectively, which is rate-determining for the entire process. Unraveling these mechanistic intricacies may help in the understanding of the natural evolution of enzymes for diverse substrates and establish the enzyme structure-function relationship.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

An Integrated Analytical Approach for Screening Functional Post-Translational Modification Sites in Metabolic Enzymes.

Post-translational modifications (PTMs) are pivotal in the orchestration of diverse physiological and pathological processes. Despite this, the identification of functional PTM sites within the vast amount of data remains challenging. Conventionally, those PTM sites are discerned through labor-intensive and time-consuming experiments. Here, we developed an integrated analytical approach for the identification of functional PTM sites on metabolic enzymes via a screening process. Through gene ontology (GO) analysis, we identified 269 enzymes with lysine 2-hydroxyisobutyrylation (Khib) from our proteomics data set of Escherichia coli. The first round of screening was performed based on the enzyme structures/predicted structures using the TM-score engineer, a tool designed to evaluate the impact of PTM on the protein structure. Subsequently, we examined the influence of Khib on the enzyme-substrate interactions through both static and dynamic analyses, molecular docking, and molecular dynamics simulation. Ultimately, we identified NfsB K181hib and ThiF K83hib as potential functional sites. This work has established a novel analytical approach for the identification of functional protein PTM sites, thereby contributing to the understanding of Khib functions.

Molecular modelling of DNA cytosine methylation

Epigenetic modifications are significant processes controlling gene expression. Since their discovery by C. H. Waddington, several multisubject research proceeded and allowed investigation of how biochemical regulations can influence gene transcription. The main epigenetic modification is DNA methylation. This catalytic process of attaching methyl groups to nucleobases (mostly cytosine) is fundamental for the regulation of gene expression and chromatin accessibility. Methylated parts of the genome, which are mostly gene promoters and enhancers, are silenced due to specific biological mechanisms. Scientists postulate that aberrant DNA methylation and other epigenetic modifications can contribute to the development of diverse pathologies such as Alzheimer's disease, epilepsy, Schizophrenia, tumorigenesis, and atherosclerosis. Due to these discoveries, it is important to characterize in detail the mechanism of epigenetic modifications to develop new possible ways of treatment. In this work, we presented complex studies of DNA methylation mechanism including dynamic and energetic analysis of the reaction system. A cluster chemical approach was used to model the active site of enzyme catalyzing methylation - DNMT1 (DNA methyltransferase 1). The created model was subjected to quantum chemical calculations. Geometry optimization was performed with ADF software, providing an energetic characterization of the studied process. Dynamic properties of the enzyme model were described due to molecular dynamics (MD) procedures performed with CP2K software. MD calculations were focused on the observation and description of proton transfer between glutamate acid and cytosine, which is considered the main enzyme-substrate interaction leading to the creation of 5-methylcytosine 5mC (product of the studied reaction) and covalent bonding between enzymes cysteine and nucleobase. This research provides a comprehensive understanding of the DNA methylation mechanism and may contribute to the invention of new drugs and modern ways of treatment.

The effect of buffer pH on enzyme activity

This study investigates the effect of pH on enzyme-catalyzed activity, with an emphasis on the involvement of wheat germ acid phosphatase in dephosphorylation processes. Temperature, pH, and enzyme concentration all have a substantial impact on enzyme activity and substrate interaction. Wheat germ acid phosphatase was chosen for this work. The activity of the enzyme in dephosphorylating p-nitrophenyl phosphate (p-NPP) into p-nitrophenol (p-NP) was evaluated using a stopped assay approach. The reaction was stopped by introducing sodium hydroxide, which ionises p-NP and produces a yellow anion visible at 405 nm. The experiment measured product production in nmol per minute and included three sections: Created a standard for the amount of p-NP with its absorbance at 405 nm; Found the ideal incubation period for maximal product generation by wheat germ acid phosphatase; Evaluated the effect of pH on enzyme activity measuring the first reaction velocity in various pH settings. The results showed that when the buffer pH diverged from the ideal value of 5.5, catalysis rates reduced dramatically, implying that high pH levels may modify the protein structure of the enzyme and reduce its catalytic effectiveness. This work emphasises the importance of pH in enzymatic activity and its potential impacts on enzyme structural integrity.

Machine Learning to Predict Enzyme-Substrate Interactions in Elucidation of Synthesis Pathways: A Review.

Enzyme-substrate interactions play a fundamental role in elucidating synthesis pathways and synthetic biology, as they allow for the understanding of important aspects of a reaction. Establishing the interaction experimentally is a slow and costly process, which is why this problem has been addressed using computational methods such as molecular dynamics, molecular docking, and Monte Carlo simulations. Nevertheless, this type of method tends to be computationally slow when dealing with a large search space. Therefore, in recent years, methods based on artificial intelligence, such as support vector machines, neural networks, or decision trees, have been implemented, significantly reducing the computing time and covering vast search spaces. These methods significantly reduce the computation time and cover broad search spaces, rapidly reducing the number of interacting candidates, as they allow repetitive processes to be automated and patterns to be extracted, are adaptable, and have the capacity to handle large amounts of data. This article analyzes these artificial intelligence-based approaches, presenting their common structure, advantages, disadvantages, limitations, challenges, and future perspectives.

A proximity labeling-based orthogonal trap strategy identifies HDAC8 promotes cell motility by modulating cortactin acetylation

It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates invivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate invivo. Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.

Development of Direct Electron Transfer-Type Extended Gate Field Effect Transistor Enzymatic Sensors for Metabolite Detection.

In this work, direct electron transfer (DET)-type extended gate field effect transistor (EGFET) enzymatic sensors were developed by employing DET-type or quasi-DET-type enzymes to detect glucose or lactate in both 100 mM potassium phosphate buffer and artificial sweat. The system employed either a DET-type glucose dehydrogenase or a quasi-DET-type lactate oxidase, the latter of which was a mutant enzyme with suppressed oxidase activity and modified with amine-reactive phenazine ethosulfate. These enzymes were immobilized on the extended gate electrodes. Changes in the measured transistor drain current (ID) resulting from changes to the working electrode junction potential (φ) were observed as glucose and lactate concentrations were varied. Calibration curves were generated for both absolute measured ID and ΔID (normalized to a blank solution containing no substrate) to account for variations in enzyme immobilization and conjugation to the mediator and variations in reference electrode potential. This work resulted in a limit of detection of 53.9 μM (based on ID) for glucose and 2.12 mM (based on ID) for lactate, respectively. The DET-type and Quasi-DET-type EGFET enzymatic sensor was then modeled using the case of the lactate sensor as an equivalent circuit to validate the principle of sensor operation being driven through OCP changes caused by the substrate-enzyme interaction. The model showed slight deviation from collected empirical data with 7.3% error for the slope and 8.6% error for the y-intercept.

Structural determinants of cold activity and glucose tolerance of a family 1 glycoside hydrolase (GH1) from Antarctic Marinomonas sp. ef1.

Cold-active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here-reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298-331), whereas the dynamic behavior of loops flanking the active site (residues 47-61 and 407-413) may favor enzyme-substrate interactions at the optimal temperature of catalysis (Topt ) by tethering together protein regions lining the active site. Stapling of the N-terminus onto the surface of the β-barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a "gatekeeping" hydrophobic residue (Leu178), located at the entrance of the active site.

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases

Hyperthermophilic (‘superheat-loving’) archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.Graphical

Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function.

Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.

Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Exploiting the Carboxylate-Binding Pocket of β-Lactamase Enzymes Using a Focused DNA-Encoded Chemical Library.

β-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important β-lactam class of antibiotics. The OXA-48 and NDM-1 β-lactamases cause resistance to the last-resort β-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel β-lactamase inhibitors. We exploited the β-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all β-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-β-lactam pharmacophores for the development of β-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Bio-Durability and Engineering Characteristics of Heat-Treated Poplar Wood

The aim of this study was to evaluate the effect of brown rot fungus Coniophora puteana activity on physical and mechanical properties as well as biological resistance of heat-treated poplar wood. Two poplar wood species (Populus deltoids and Populus nigra) were heat-treated by thermo-wood (Thermo-D) method. Control and heat-treated specimens were exposed to brown rot fungus C. puteana for 16 weeks. Physical and mechanical characteristics of specimens including density, compression strength parallel to the grain and impact strength were evaluated before and after exposure to fungus. Mass loss of specimens caused by fungal activity (MLF) was also calculated. In addition, the effect of thermal modification on laccase production by C. puteana was assayed. The highest mass loss due to fungal deterioration was observed in control specimens, coinciding with the highest substrate-enzyme interactions and constant decrease in detectable laccase levels. According to the results, thermal modification can be used effectively to protect poplar wood against brown rot fungus attack.

Degradation kinetics of medium chain length Polyhydroxyalkanoate degrading enzyme: a quartz crystal microbalance study.

This study investigates the enzymatic degradation processes of different classes of polyhydroxyalkanoates (PHAs), a group of biopolymers naturally synthesized by various microorganisms. Medium chain length PHAs (mcl-PHAs) are distinguished biopolymers due to their biodegradability and diverse material properties. Using quartz crystal microbalance measurements as a valuable tool for accurate real-time monitoring of the enzymatic degradation process, the research provides detailed kinetic data, describing the interaction between enzymes and substrates during the enzymatic degradation process. Thin films of poly-3-hydroxybutyrate (PHB) and polyhydroxyoctanoate copolymer (PHO), containing molar fractions of about 84% 3-hydroxyoctanoate and 16% 3-hydroxyhexanoate, were exposed to scl-depolymerases from Pseudomonas lemoignei LMG 2207 and recombinant mcl-depolymerase produced in Escherichia coli DH5α harboring the plasmid pMAD8, respectively. Analyses based on a heterogeneous kinetic model for the polymer degradation indicated a six-fold stronger adsorption equilibrium constant of mcl-depolymerase to PHO. Conversely, the degradation rate constant was approximately twice as high for scl-depolymerases acting on PHB. Finally, the study highlights the differences in enzyme-substrate interactions and degradation mechanisms between the investigated scl- and mcl-PHAs.

Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase GmUGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.

Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon‑selenium (C–Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C–Se bond formation.